Mogrol stimulates G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5) and insulin secretion from pancreatic β-cells and alleviates hyperglycemia in mice

Target identification is a crucial step in elucidating the mechanisms by which functional food components exert their functions. Here, we identified the G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) as a target of the triterpenoid mogrol, a class of aglycone mogroside derivative from Siraitia grosvenorii. Mogrol, but not mogrosides, activated cAMP-response element-mediated transcription in a TGR5-dependent manner. Additionally, mogrol selectively activated TGR5 but not the other bile acid-responsive receptors (i.e., farnesoid X receptor, vitamin D receptor, or muscarinic acetylcholine receptor M3). Several amino acids in TGR5 (L71A2.60, W75AECL1, Q77AECL1, R80AECL1, Y89A3.29, F161AECL2, L166A5.39, Y240A6.51, S247A6.58, Y251A6.62, L262A7.35, and L266A7.39) were found to be important for mogrol-induced activation. Mogrol activated insulin secretion under low-glucose conditions in INS-1 pancreatic β-cells, which can be inhibited by a TGR5 inhibitor. Similar effects of mogrol on insulin secretion were observed in the isolated mouse islets. Mogrol administration partially but significantly alleviated hyperglycemia in KKAy diabetic mice by increasing the insulin levels without affecting the β-cell mass or pancreatic insulin content. These results suggest that mogrol stimulates insulin secretion and alleviates hyperglycemia by acting as a TGR5 agonist.


Simulated docking of mogrol to TGR5
To identify TGR5 residues involved in mogrol binding, we computationally docked mogrol with TGR5 from the semisynthetic bile acid INT-777-bound TGR5 structure 8 using GNINA.In 20 docking poses, GNINA pose scores ranged from 0.806 to 0.584.Vina scores ranged from − 10.8 to − 8.77 kcal mol −1 .The previously reported cryo-EM structure (PDB code: 7CFN) shows that TGR5-bound INT-777 inserts its tetracyclic moiety into the receptor interior 8 .Based on the structural similarity between mogrol and INT-777, we focused on the 12th docking pose (GNINA pose score: 0.64; Vina score: − 9.52 kcal mol −1 ) in which the position of the tetracyclic moiety is close to that of INT-777 in the cryo-EM structure (Fig. 3a).In the focused pose, six residues (L74 2.63 , Y89 3.29  F96 3.36 , F161 ECL2 , L262 7.35 , and L266 7.39 ) were involved in van der Waals (vdW) interactions with mogrol, and the side chain of S270 7.43 and the main chain of A250 6.61 were involved in hydrogen bonds to mogrol (Fig. 3b and c).

Mogrol increased insulin secretion by activating TGR5 in β-cells
Activation of TGR5 promotes insulin secretion by pancreatic β-cells 16 .We thus examined the effects of mogrol on insulin secretion by INS-1 β-cells.INS-1 cells were stimulated by mogrol for 1 h, and secreted insulin was determined by ELISA.As shown in Fig. 5a, mogrol significantly stimulated insulin secretion under low-glucose, but not high-glucose, conditions.The enhancement was inhibited by the TGR5 inhibitor SBI-115.(Fig. 5b).Cell viability and intracellular insulin content were not affected by the tested concentrations of mogrol (Fig. 5c and d).
In addition, the stimulatory effects of mogrol on insulin secretion under low-glucose conditions were observed in the isolated mouse islets (Fig. 5e).

Mogrol increased plasma insulin concentrations and alleviated hyperglycemia in diabetic KKAy mice
Because mogrol stimulates insulin secretion and TGR5 activation has been reported to repress hyperglycemia 34 , we examined the effects of mogrol on blood glucose levels in the KKAy diabetic mouse model.KKAy diabetic mice were divided into three groups (i.e., a vehicle control, 0.01% mogrol, and 0.05% mogrol) and treated under these conditions for 5 weeks.C57BL/6 J mice were used as a non-diabetic control group.Although the body weight of KKAy mice was higher than that of C57BL/6 J mice, mogrol supplementation exerted no effects on body weight or feeding efficiency among KKAy mice (Fig. 6a and b).Liver and plasma lipid levels in KKAy mice were not significantly affected by mogrol intake (Table 1), but plasma triglyceride levels tended to decrease (p = 0.08 at 0.01% mogrol group).Both intraperitoneal and oral glucose tolerance tests showed that mogrol intake dosedependently lowered blood glucose concentrations compared with those of KKAy diabetic control mice, whose blood glucose concentrations were elevated compared to C57BL/6 J non-diabetic control mice (Fig. 6c and  d).Mogrol intake did not affect insulin sensitivity in KKAy mice (Fig. 6e), but did dose-dependently increase plasma insulin concentrations (Fig. 6f).Pancreatic β-cell mass (Fig. 6g and h) and pancreatic insulin protein levels (Fig. 6i) were not affected by the administration of mogrol.

Discussion
Recent studies have revealed the physiological effects of mogrol, including as anti-inflammatory and anti-osteoporotic activities in mice [3][4][5][6] , but to date, a molecular target of mogrol has not been elucidated.In this study, we showed that mogrol selectively activates TGR5, while other bile acid receptors, including FXR, VDR, and CHRM3, do not 12 .Docking simulation analysis and evaluation of activation using TGR5 mutants indicate that mogrol acts as an agonist for TGR5.Furthermore, we have demonstrated that mogrol functions as an insulin secretagogue by activating TGR5 in pancreatic β-cells and alleviates hyperglycemia in mice.Mogrol, but not mogrosides, activated TGR5.Activation of TGR5 by mogrol but not by DCA was abolished in variants with the alanine scanning mutations W75A ECL1 , R80A ECL1 , S247A 6.58 , L262A 7.35 , and L266A 7.39 .In contrast, activation of TGR5 by mogrol and DCA was abolished by the mutations L71A 2.60 , Q77A ECL1 , Y89A 3.29 , F161A ECL2 , L166A 5.39 , Y240 6.51 , and Y251A 6.62 .Our results indicating that mutations L71 2.60 , Y89 3.29 , F161 ECL2 , L166 5.39 , Y240 6.51 , S247 6.58 , Y251A 6.62 , L262 7.35 , and L266 7.39 reduce mogrol potency were similar to results obtained with INT-777 8 .In addition, our observation that Y89A 3.29 in TGR5 reduced sensitivity to a bile acid was consistent with a previous study 35 , whereas decreased DCA sensitivity was ameliorated with the S270A 7.43 mutation relative to the Y89A 3.29 mutation.W75 ECL1 acts as an active site at lid 36 , and mutation at this site can selectively increase DCA potency or decrease the potency of mogrol and P395 8 but does not affect 23H or litocholic acid 36 .Sasaki et al. 21have shown that limonoid compounds (the most abundant triterpenoids in citrus fruits) nomilin and obacunone activate TGR5, whereas in the triple mutants Q77R ECL1 /R80Q ECL1 /Y89H 3.29 and Q77A ECL1 / R80A ECL1 /Y89A 3.29 activation by nomilin and/or obacunone, but not taurolithocholic acid, was abolished 21 .These results suggest that mutations in Q77 ECL1 , R80 ECL1 , and Y89 3.29 exert a negligible effect on overall structure of TGR5.F96A 3.36 compromised the potency of the TGR5 agonist 23H but not that of litocholic acid 36 .Our result www.nature.com/scientificreports/with the F96A mutant was similar to the results with litocholic acid.Overall, these results indicate that mogrol binds directly to and acts as an agonist of TGR5.TGR5 was not activated by mogrosides, glycosides of mogrol, whereas the glycosides of quinovic acid 18 and glycyrrhizic acid (a glycoside of glycyrrhetinic acid) 20 can activate this receptor.The hypoglycemic effect of ginsenoside Ro, a glycoside of the TGR5 agonist oleanolic acid, was attenuated in TGR5-deficient mice 37 , whereas it remains unclear whether ginsenoside Ro is itself without deglycosidation or has a direct effect on β-cells.Mogrol-stimulated insulin secretion was inhibited by the TGR5 inhibitor, suggesting that mogrol stimulates insulin secretion by activating TGR5.Our results support the hypothesis that TGR5 activation stimulates insulin secretion in pancreatic β-cells 16 .Although CHRM3 is expressed in β-cells and contributes to insulin secretion 38 , mogrol did not affect CHRM3 activation.To our knowledge, the ability of terpenoids and related compounds to modulate insulin secretion by activating TGR5 has not previously been experimentally demonstrated.
Mogrol increased the insulin secretion under low-glucose conditions in INS-1 cells and isolated mouse islets.Kumar et al. 16 showed that TGR5 couples with Gαs, but not Gαq, Gαi, and Gα 12/13 , in MIN6 β-cells and that the activation of TGR5 by oleanolic acid (10 μM), INT-777, and lithocholic acid enhances insulin secretion both under low-and high-glucose concentrations in MIN6 and human β-cells.In addition, the intracellular calcium levels increased at low-glucose concentrations.Although overdosage of insulin secretagogues should be noted in relation to hypoglycemia, to our knowledge, the occurrence of hypoglycemia by TGR agonists has not been reported.Tauroursodeoxycholic acid and oleanolic acid (1 μM) increases the glucose-stimulated insulin secretion from the mouse or human islets under high-glucose, but not low-glucose, concentrations 15,39 .These results suggest that different ligands may alter the binding mode of the TGR5 receptor and activate different signals, particularly intracellular calcium signaling.Different concentrations of compound could affect cellular signaling in complex ways.The reduced insulinotropic effect of mogrol under high-glucose concentrations may be due to its AMPK activity-promoting effect 3,7,40 as AMPK activation has a negative impact on insulin secretion 41 , although this remains controversial 42 .
In contrast to some TGR5 agonists 37,43,44 , mogrol did not affect the body weight or insulin sensitivity in mice.Muscle-specific TGR5 transgenic mice exhibit increased glucose tolerance and a slight improvement in insulin sensitivity (P = 0.066), without any change in their body weight 45 .Therefore, the effects of muscle TGR5 activation on insulin sensitivity may be limited.The bioavailability, tissue distribution, and tissue accumulation of compounds may have caused these differences.
Dietary supplementation (0.4%) with crude extract of Siraitia grosvenorii (SG-ex) prevented impaired insulin secretion and glucose intolerance in Goto-Kakizaki rats, presumably due to decreased oxidative stress in the pancreas 46 .By contrast, a single administration of a mogroside-rich extract of Siraitia grosvenorii (SG-gly) can alleviate a rise in blood glucose concentrations after maltose but not glucose loading in rats 47 .SG-gly contents in SG-ex are low.Thus, anti-oxidative components other than SG-gly in SG-ex seem to be responsible for the observed anti-hyperglycemic effects.Therefore, compounds from Siraitia grosvenorii have several targets by which they may ameliorate hyperglycemia (i.e., SG-gly, maltase inhibitory activity; SG-ex, anti-oxidative activity in the pancreas; mogrol, insulin secretory activity).
In this study, we have demonstrated that TGR5 is a target of mogrol and have identified several amino acids in TGR5 that are key to mogrol binding.Mogrol activated CRE-mediated transcriptional activation in TGR5expressing cells.By contrast, in 3T3-L1 preadipocytes, mogrol suppresses CRE-mediated CREB activity 7 .This inconsistency may be due to the existence of mogrol targets other than TGR5.In the present study, we showed that mogrol activated TGR5 and insulin release from pancreatic β-cells, alleviating hyperglycemia at least partly in diabetic KKAy mice.Further studies to identify other mogrol targets could uncover different mechanisms underlying mogrol mediated physiological effects.A limitation of this study is that the TGR5-mediated function of mogrol lacks in vivo experiment.Further mechanistic studies of mogrol using gene knockout animals are warranted.Mogrosides are natural sweeteners that can be used as sugar alternatives.After ingestion, mogrol is produced by the deglycosylation of mogrosides in the intestinal tract and subsequently absorbed by the body 1,2 .Therefore, mogroside intake, in addition to direct mogrol supplementation, may contribute to the prevention of T2DM by exerting effects other than the reduction of sugar intake.

Materials
Mogrol and mogrosides were prepared as described previously 7 .Their structures are shown in Fig. 1.These compounds were purified to at least 96% via HPLC and dissolved in DMSO.

Cell viability assay
INS-1 cells grown in a 48-well plate were cultured with 50 μM mogrol for 24 h, followed by incubation with 5% alamarBlue (Bio-Rad, Hercules, CA, USA) for 4 h under light shielding.Fluorescence was measured at excitation and emission wavelengths of 544 and 590 nm, respectively, using Fluoroscan Ascent FL (Labsystems, Helsinki, Finland).

Insulin secretion assay and measurement of insulin levels in INS-1 cells
The amounts of insulin secreted by INS-1 cells were determined as previously described 53 .Briefly, INS-1 cells seeded in a 48-well plate were incubated with the Krebs-Ringer bicarbonate buffer containing 0.1% bovine serum albumin and glucose (2.8 or 16.7 mM) in the presence or absence of 50 μM mogrol for 1 h.Cells were pretreated with the TGR5 inhibitor, SBI-115, for 40 min, followed by mogrol treatment.INS-1 cells grown in a 48-well plate were cultured with 50 μM mogrol for 24 h.Cell lysates were prepared using a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40, and 2 mM EDTA).The amounts of secreted insulin and intracellular insulin were determined using sandwich ELISA as previously described 53 .Bovine insulin was used as the standard.

Insulin secretion assay using isolated mouse islets
Pancreatic islets were isolated from 8-9-week-old ICR mice (Kiwa Laboratory Animals, Wakayama, Japan) as reported previously 53 , with minor modification.Briefly, after collagenase digestion, the pancreas was fractionated via Ficoll-diatrizoate density gradient centrifugation.Mouse islets (3-4 islets/tube) were incubated with the Krebs-Ringer bicarbonate buffer containing 0.1% bovine serum albumin with a basal (2.8 mM) or stimulatory (16.7 mM) level of glucose for 1 h in the presence of mogrol.Secreted and intracellular insulin concentrations were measured using ELISA, as previously described 53 .Finally, the ratio of secreted to intracellular insulin was calculated.

Structure-based docking simulation
Coordinates from a structure of human TGR5-G s complex with bound bile acid derivative INT-777, solved by cryo-electron microscopy (cryo-EM) (Protein Data Bank [PDB] code: 7CFN) 8 were used for our docking studies examining TGR5-mogrol binding.A three-dimensional structure of mogrol was obtained from PubChem (CID: 14,525,327).Hydrogen atoms and missing heavy atoms were added to the experimental TGR5 structure using Protein Repair and Analysis Server 55 .In silico docking of the mogrol molecule with the TGR5 structure was performed using GNINA 56 version 1.0.2(derivative of SMINA 57 and AutoDock Vina 58,59 that supports convolutional neural network scoring).The docking box in the simulation was automatically set up with reference to the INT-777 coordinates in 7CFN.During docking simulations, the rotational bands of the small molecule were explicitly considered to be flexible, and the protein was treated as a rigid body.TGR5-mogrol interactions in the docking results were analyzed and detected using Protein-Ligand Interaction Profiler 60,61 version 2.2.2.

Animals
Male 4-week-old C57BL/6 J and KKAy mice were obtained from CLEA Japan (Tokyo, Japan) and individually housed (cage size: 136 × 208 × 115 mm; bedding material: clean chip, CLEA Japan).Additionally, male 8-week-old ICR mice were obtained from Kiwa Laboratory Animals and housed in a group (cage size: 225 × 338 × 140 mm; bedding material: clean chip) under conventional conditions with controlled temperatures (23 ± 3 °C), a 12/12-h light/dark cycle (light period starting at 08:00 AM), and ad libitum access to standard food (CE-2, CLEA Japan) and water.After acclimation for one week, C57BL/6 J mice (the non-diabetic control group, n = 6, 20.4 ± 0.2 g) continued to receive the standard diet for 5 weeks, while KKAy diabetic mice were divided into three groups (n = 8-9, diabetic control group, 28.4 ± 0.2 g; 0.01% mogrol group, 28.7 ± 0.4 g; 0.05% mogrol group, 28.5 ± 0.7 g), with each group having a similar body weight distribution and fed a high-fat diet (HFD) 62 for 5 weeks.The KKAy diabetic control group was fed HFD alone, and the other groups were fed HFD containing 0.01 or 0.05% (w/w) mogrol.For the intraperitoneal glucose tolerance test (IPGTT), oral glucose tolerance test (OGTT), and insulin tolerance test (ITT), the mice fasted for 6 h (from 09:00 AM), then 2 g/kg glucose or 1 U bovine insulin were intraperitoneally or orally administered.Blood was collected from the tail vein, and glucose levels were measured using a Stat Strip Express Glucose/Ketone meter (Nova Biomedical, Waltham, MA, USA).These tolerance tests were performed in animal care room.After 4 h of fasting (from 09:00 AM), mice were kept unconscious under isoflurane anesthesia (5% for introduction and 2% for maintenance, SN-487, Shinano Seisakusho, Tokyo, Japan) and euthanized by exsanguination from inferior vena cava, and blood and organs were harvested for further analysis.Four groups of mice were treated and dissected alternately.After acclimation for one week, ICR mice (n = 2) were euthanized via cervical dislocation, and the pancreatic islets were isolated.All animal experiments were approved by the Animal Care and Use Committee of the Osaka Metropolitan University (Nos.22-17 and 23-132) and were performed in compliance with its guidelines and the ARRIVE guidelines.

Immunohistochemistry and measurement of pancreatic insulin levels
Pancreatic β-cell mass was determined as previously described 63 .Briefly, formalin-fixed paraffin-embedded pancreatic tissue sections were prepared.The sections were mixed with an anti-insulin antibody (D6C4, Hytest, Turku, Finland), Histofine Simple Stain mouse MAX-PO (Nichirei, Tokyo, Japan), and DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA), followed by counterstaining with hematoxylin.The tissue sections were observed under a light microscope (BZ-X810, Keyence, Osaka, Japan), and the staining intensity was determined using the Image Pro software (ver.10, Media Cybernetics, Silver Spring, MD, UDA).β-cell mass was calculated as follows: ratio of β-cell area to total pancreatic area × pancreatic weight/body weight.To determine the pancreatic insulin content, the pancreas were homogenized in acid-ethanol (1.5% HCl in 70% ethanol).After neutralization with 1 M Tris-HCl (pH 7.5), insulin concentrations were determined using ELISA, as previously described 53 , and normalized to the pancreatic protein content.

Measurement of insulin, triglyceride, cholesterol, and free fatty acid levels
Plasma insulin levels were determined using an Ultra Sensitive Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science Inc., Kanagawa, Japan).Lipids were extracted from the liver by the Folch's method.Liver and plasma triglyceride, total cholesterol, and free fatty acid (FFA) levels were determined with a triglyceride E-test (Fujifilm Wako, Osaka, Japan), a cholesterol E-test (Fujifilm Wako), and a NEFA C-test (Fujifilm Wako), respectively.Measurements were performed single or duplicate.

Figure 5 .Figure 6 .
Figure 5. Mogrol activated insulin secretion in INS-1 β-cells and isolated mouse islets.(a and b) Insulin secretion from INS-1 cells in the presence of 50 μM mogrol and/or 10 μM SBI-115 under low (2.8 mM; a and b) and high (16.7 mM; a) glucose conditions.Amounts of extracellular insulin were determined by ELISA.(c and d) Cell viability or intracellular insulin content after incubation with 50 μM mogrol for 24 h.(e) Insulin secretion from the isolated mouse islets in the presence of 50 μM mogrol under low (2.8 mM) and high (16.7 mM) glucose conditions.Amounts of extracellular and intracellular insulin were determined using ELISA, and the ratio of secreted to intracellular insulin was calculated.Data are expressed as means ± SEM (n = 4-5, for insulin secretion assay in INS-1 cells; n = 4, for cell viability assay in INS-1 cells; n = 8, for measurement of insulin content in INS-1 cells, n = 8, for insulin secretion assay in isolated mouse islets).Asterisks indicate statistically significant differences compared with relevant controls (two-way analysis of variance with Bonferroni's post hoc tests (a, b, and e) or Student's t-tests (c and d), *p < 0.05, **p < 0.01, ***p < 0.001).TGR5, G-protein-coupled bile acid receptor 1/Takeda G-protein receptor 5.
https://doi.org/10.1038/s41598-024-53380-xwww.nature.com/scientificreports/StatisticalanalysesData were analyzed by two-tailed Student's t-test or one-way analysis of variance with Tukey-Kramer's or Dunnett's post hoc tests using JMP statistical software version 8.0.1 (SAS Institute, Cary, NC, USA).Two-way analysis of variance with Bonferroni's post hoc test was performed using GraphPad Prism statistical software version 5 (GraphPad Software Inc., San Diego, CA, USA).Data are displayed as means ± SEM, and a p-value < 0.05 was considered to indicate a statistically significant difference between groups.